GSM7712421: Single B cell sequencing (scBCR-seq), LuS_mRBD_B; Mus musculus; RNA-Seq
Single-cell suspensions were obtained by grinding the lymph nodes of immunized mice. The cells from each group of mice were pooled together. Antibodies used for staining were anti-mouse GL-7-FITC, CD138-PE, CD38-PE/Cy7, CD93-APC, B220-BV421 and IgD-BV510. All antibodies were used according to manufacturer's instructions. To stain the antigen-binding B cells, a mixture of SARS-CoV-2 RBD (Prototype), SARS-CoV-2 RBD (Beta), RaTG13 RBD, RshSTT182 RBD, LyRa11 RBD, RmYN02 RBD were used as baits by biotinylation via Biotin-Protein Ligase/BirA Enzyme (GeneCopoeia). BV711-Streptavidin was used to probe the biotinylated RBD. Antigen-specific BGC gated as GL-7+, B220high, CD38low, IgD-, CD93-, CD138- and RBD+ were sorted by BD FACS Aria II flow cytometer (BD Biosciences). Antigen-specific B cells were sorted by flow cytometry and then subjected for high-throughput single-cell V(D)J sequencing with 10× Genomics platform. Brefly, after FACS sorting, cells were centrifuged, resuspended in 3% FBS (Sigma-Aldrich)/phosphate buffer solution (Thermo Fisher Scientific) and subjected to cell quality control using a cell counter. The cell viability of processed B cells was greater than 70%. After determining cell density, the single-cell library was constructed using a 10× Chromium Single Cell 5' Library and Gel Bead Kit following the manufacturer's user guide. Cells were injected into three channels, aiming to achieve around 1,000-3,000 cells per channel. Gel beads-in-emulsion (GEMs) were formed in channels of a chip in the 10× Chromium instrument, and then collected in an Eppendorf plate for the GEM-reverse transcription (GEM-RT) reaction. After GEM clean up, GEM-RT products were subjected to two rounds of 9 and 15 PCR cycles using custom primers, followed by SPRI select (Beckman Coulter) bead clean up. Single-cell BCR V(D)J libraries were prepared following the manufacturer's user guide, profiled using the Bioanalyzer High Sensitivity DNA kit (Agilent Technologies) and quantified with a Kapa Library Quantification Kit (Kapa Biosystems). Libraries were sequenced by paired-end sequencing (150-bp) on an Illumina NovaSeq (Illumina).